1. Field of the Invention
The invention relates to a universal and greatly simplified method as well as a formulation for isolating a nucleic acid from a variety of starting materials containing nucleic acids which both results a very high quality and high yields of the isolated nucleic acid.
2. Discussion of the Background
Under conventional conditions the isolation of DNA from cells and tissues is carried out such that the starting materials containing the nucleic acids are digested under highly denaturing and reducing conditions, in part with the use of protein-degrading enzymes. The released nucleic acid fraction is purified in phenol/chloroform extraction stages and the nucleic acids are isolated by dialysis or ethanol precipitation from the aqueous phase (Sambrook, J., Fritsch, E. F. und Maniatis, T., 1989, CSH, “Molecular Cloning”).
These conventional methods for the isolation of nucleic acids from cells and especially from tissues are very time consuming (in part longer than 48 h), require considerable apparative expenditure and moreover are not realizable under field conditions. In addition, such methods are hazardous to health and environment due to the chemicals used in amounts that are not inconsiderable, such as phenol and chloroform.
Different alternative methods for the isolation of nucleic acids from various biological starting materials allow the elaborate and health-damaging phenol/chloroform extraction of nucleic acids to be circumvented and a reduction in time expenditure to be achieved.
All of these methods are based on a method for the preparative and analytical purification of DNA fragments from agarose gels developed and described for the first time by Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA, 1979, 76, 615-619). The method combines the dissociation in a saturated solution of a chaotropic salt (NaI) of the agarose containing the bands of the DNA to be isolated with binding of the DNA to glass particles. The DNA fixed to the glass particles is then washed with a wash solution (20 mM Tris HCl [pH 7.2]; 200 mM NaCl; 2 mM EDTA; 50% v/v ethanol) and then separated from the support particles.
Until now, this method has undergone a series of modifications and is currently used for different methods for the extraction and purification of nucleic acids from various sources (Marko, M. A., Chipperfield, R. und Birnboim, H. G., 1982, Anal. Biochem., 121, 382 387).
In addition, a plurality of reagent systems exists world-wide today, predominantly for the purification of DNA fragments from agarose gels and for the isolation of plasmid DNA from bacterial lysates, and also for the isolation of longer chain nucleic acids (genomic DNA, cellular total RNA) from blood, tissues or cell cultures.
All these commercially available kits are based on the well-known principle of binding nucleic acids to mineral supports in the presence of solutions of different chaotropic salts and use suspensions of finely-milled glass powder (e.g. Glasmilk, BIO 101, La Jolla, Calif.), diatomaceous earths (Sigma) or silica gels (Diagen, DE 41 39 664 A1) as support material.
A method for the isolation of nucleic acids practicable for a number of different applications proposed in U.S. Pat. No. 5,234,809. A method is described therein for the isolation of nucleic acids from starting materials containing nucleic acids, whereby the starting material is incubated with a chaotropic buffer and a DNA-binding solid phase. The chaotropic buffer carries out both the lysis of the starting material as well as the binding of the nucleic acids to the solid phase. The method is well suited for the isolation of nucleic acids from small amounts of sample and finds practical use particularly in the area of the isolation of viral nucleic acids.
Specific modifications of these methods concern the use of novel support materials which have applicative advantages for particular problems (WO-A 95/34569).
More recent patent applications disclose that so-called antichaotropic salts can be used very efficiently and successfully as components of lysis/binding buffer systems for the adsorption of nucleic acids to silicate materials known and used by the person skilled in the art (EP 1 135 479 A). The advantage of this method is that by circumvention of the use of chaotropic salts a clearly lower hazard to health is posed by the extraction system. However, a draw-back is that high salt concentrations (>1.5 M) are required in the lysis buffer for an efficient isolation of nucleic acids from a complex biological sample especially with respect to a highest possible nucleic acid recovery. Thus, EP 1 135 479 A discloses that the lysis buffers used contain salt concentrations of between 1.5 M-3 M.
A method is described in the unexamined application DE 43 21 904 A in which an efficient isolation of nucleic acids is possible with a combination of chaotropic high salt buffers and alcoholic components. The lysis buffers disclosed in DE 43 21 904 A always contain salt concentrations of 4 M-8 M, guanidine hydrochloride, guanidine thiocyanate or potassium iodide in particular are used as salts. It is known that these salts bring about lysis of the starting material as well as potent inactivation of nucleolytic enzymes. The addition of an alcohol is carried out after lysis of the starting material. The patent document discloses that the addition of the alcoholic component to the high salt lysis buffer mediates a highly efficient binding of the nucleic acids to the silicate filter material employed. The disadvantage of the use of lysis buffers with high ion strength chaotropic salts is always the restricted and also inefficient use of additional proteolytic enzymes for an effective digestion of complex biological samples, for these enzymes are themselves damaged by the protein-denaturing action of chaotropic buffers. Furthermore, extensive wash stages are needed subsequently to remove the high salt concentrations from the adsorption material employed. It is known to the person skilled in the art that chaotropic salts exert a high inhibitory action on a number of down-stream applications.
A further possibility for the isolation of nucleic acids is disclosed in utility patent application DE 20 20 7793 U. The method includes the binding of nucleic acids to clay minerals in the presence of a salt of a polyvalent cation. The method is considered to be based on the formation of a so-called cation bridge between the nucleic acids to be isolated and the clay minerals. The desorption of the nucleic acids to be isolated does not take place with water or a low salt buffer as with other known methods but with an elution agent which is at least one complexing agent that is specific for the polyvalent cation in the binding buffer. The disadvantage of the invention is in particular that the complexing components (EDTA; EGTA etc.) as part of the elution buffer often greatly inhibit a number of downstream applications. Although the method for the purification of nucleic acids as described in the utility patent specification ought to be feasible without the use of chaotropic salts, a series of buffers of the embodiment examples always contain chaotropic salts (e.g. guanidine salts). This concerns all embodiment examples that describe the isolation of nucleic acids from complex biological samples. It thus appears that the isolation of nucleic acids from complex samples cannot be realized in the absence of chaotropic salts.
DE 198 56 064 A1, DE 102 53 351 A1, DE 699 08 795 T2 and EP 0 796 327 B1 also belong to the background art. Methods for the isolation of nucleic acids are described therein whereby, however, no antichaotropic components are used.
The analysis of the background art illustrates quite impressively that a plurality of possibilities exists for the binding nucleic acids to solid support materials, in particular silicon-based mineral support materials, then to wash and to release once more the nucleic acids from the support material. It is also clear that a plurality of lysis buffer systems can be employed for the isolation of nucleic acids from complex biological samples. The lysis buffer systems described also always contain salt components that are essential for the necessary binding of the nucleic acids to be isolated to the respective preferred support material, whereby the chaotropic salts selected for the binding of the nucleic acids also at the same time carry out the lysis of the starting material. The salt concentrations of the lysis buffers employed always lie in the high-salt range. This also applies to the utility patent specification DE 20 20 7793 U, although as explicitly described there the binding of the nucleic acids with the use of polyvalent cations and the utilization of clay minerals should take place with low concentrations of polyvalent salts. The concentrations of the guanidinium salts used in this utility patent specification for the isolation are the high salt concentrations known to the person skilled in the art.